Congenital defects of the valves and septa of the heart are among the most common human malformations and are the leading cause of birth defect-related deaths. Understanding the molecular basis of cardiac malformations may lead to improved methods for their diagnosis, classification, and treatment. Recently, several genes have been identified that cause congenital heart defects in human families or in experimental mice. Among these is Jag-l, a ligand for the Notch signaling pathway that is deleted or mutated in Alagille syndrome and in some cases of tetralogy of Fallot. We have induced a targeted mutation in the mouse Hey1 and Hey2 genes, basic Helix-Loop-Helix (bHLH) transcription factors thought to be regulated by Notch signaling. Hey2-'- animals develop a spectrum of cardiac valvuloseptal malformations, including tetralogy of Fallot, membranous interventricular septal defect, and tricuspid atresia. The similarities between the Hey2 mutant phenotype and human malformations associated with Jag1 mutations suggest that Hey2 may regulate and/or mediate Notch signaling during mammalian cardiac development. Hey1 -/- develop strain specific defects of the aortic arches, and doubly mutant embryos die prenatal. To better understand how the lack of Hey2 protein alters pattern formation and cellular differentiation in the developing heart, we now propose to characterize the cardiac malformation of Hey2 mutant animals using molecular reagents specific for individual cell types or domains of the developing heart. In addition, we will test the hypothesis that Hey2 works in Notch signaling in the mouse embryonic heart in two ways. We will examine the expression of Jag1 and other components of the Notch signaling pathway in Hey2 mutant animals, as well as the expression of Hey2 in animals lacking Jag1, Notch1, or Notch2. Furthermore we will test for genetic interactions between the Hey2 and Jag, Notch1, or Notch2 mutant alleles. We will determine the effect of altering Hey2 expression or Notch signaling activity in the developing heart by expressing wild type and mutant forms of Hey2 and of Notch pathway components in chick embryos. Finally, we will characterize the cellular and molecular basis for the embryonic lethality of Hey1-/-; Hey2 -/- embryos. These studies will clarify the role of Hey2 in cardiac valvuloseptal morphogenesis, test the hypothesis that Hey2 functions in heart development by modifying the Notch signaling pathway, and test the hypothesis that Hey1 and Hey2 act together to regulate cardiovascular development in the mouse.